Perturbation of METTL1-mediated tRNA N7- methylguanosine modification induces senescence and aging

Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.


FigS1
FigS1.Measurement of METTL1/WDR4 level during senescence and aging.a METTL1 and WDR4 level were detected in senescence models.P represents proliferating IMR90 cells.Sen refers to senescent IMR90 cells.P+ETO stands for proliferating cells treated with ETO.b Transcript levels of METTL1, WDR4, and senescence markers were measured at various time points during senescence using RT-qPCR.c Transcription levels of Mettl1 and p16 were shown in tissues of young and old mice, n=8 mice per group (4 female and 4 male mice).dProtein level of Mettl1 and Wdr4 were shown in lung tissues of young and old mice, n=5 mice per group (2 female and 3 male mice).Data in

FigS2
FigS2.METTL1 Deficiency Accelerate Cell Senescence a METTL1 was depleted by CRISPR in IMR90 cells, senescence status was assessed by assays including western blotting(a), SA-β-gal staining(b), EdU incorporation(c).Scale bar = 100 μm, P values were calculated using a onetailed Student's t-test.d Differentially regulated SASP genes in RNA-Seq of METTL1 knockout cells were enriched using GSEA analysis.e m7G levels in total RNA and tRNA from METTL1 KO cells were analyzed using HPLC/MS.Three repeats were conducted.f Ectopic expression of METTL1 and AFPA-mut proteins, labeled with a FLAG tag at either the C-terminus or HA at the Nterminus, were detected in IMR90 cells with anti-METTL1 or tag antibodies as indicated.The left panel displays Western blotting using METTL1-FLAG construct; the right panel exhibited Western blotting using HA-METTL1 construct.g m7G modification in IMR90 cells with ectopic expression of METTL1 and AFPA-mut was analyzed using HPLC-MS.Source data are provided as a Source Data file.

FigS3a
FigS3.Depletion of Mettl1 Induces Premature Aging a Schematic diagram illustrating the construction strategy for conditional METTL1 knockout and conditional METTL1 knockin mice(a) and the experimental strategy for METTL1 knockout and conditional METTL1

FigS4a
FigS4.METTL1 Deficiency Downregulates a Group of m7G-tRNAs a The proportion of small RNAs mapping in tRNA-seq samples from senescence groups was shown.b The proportion of small RNAs mapping in tRNA-seq samples from METTL1 knockout (KO) groups was shown.c Ratios of cleavage scores in young and senescent cells were characterized.Cleavage Score Ratio = Cleavage Score (Sample 1) / Cleavage Score (Sample 2).d Differentially expressed m7G-tRNAs in control/METTL1 KO (Day 10) cells were showed.All tRNA genes of the same tRNA type were combined to calculate their transcript abundance.Source data are provided as a Source Data file.

FigS5
FigS5.Ribo-Seq Uncover Codon Usage upon Mettl1 Depletion a-c Characteristics of ribosome footprinting.(a) Distribution of the read length.(b) The percentage of P-sites falling in three possible translation reading frames for 5' UTRs, CDSs, and 3' UTRs in ribosome footprinting data.(c) The meta-

FigS6
FigS6.METTL1 Deficiency Elicits Ribotoxic Stress Response andIntegrated Stress Response a ISR and RSR response-relevant proteins were detected by Western blotting using kidney tissues from young/old mouse(a) (n=5 mice per group, 2 female and 3 male) and Mettl1 KO/ control mice(b) (n=5 mice per group, 3 female and 2 male) kidney tissues.c Validation of the effect of GCN2 and p38 inhibitors on METTL1-depleted cells was performed by western blotting to detect eIF2α and p38 phosphorylation.All WB results were performed three times, the representative result was shown.

FigS7.
FigS7.RTD is Involved in the Senescence Caused by METTL1 Deficiency a eEF1A level was detected by RT-qPCR with samples of replicative senescence, and METTL1 depleted IMR90 cells by shRNAs and sgRNAs.Three repeats were conducted, P values were calculated using a two-tailed Student's T-test.Data was presented as mean ± SEM. b Represent images of FlucDM-GFP expressing cells are showed.Source data are provided as a Source Data file.

FigS8
FigS8.SP1 Regulates METTL1 and WDR4 Expression a METTL1 level were quantified using RT-qPCR in IMR90 cells with short hairpin RNAs (shRNAs) targeting p53, RB1, and cMyc.Three biologically repeats were conducted.P values were calculated using a two-tailed Student's T-test.Data is presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.b Schematic diagram of dual luciferase reporter gene assay.Source data are provided as a Source Data file.